Fluorescein diacetate hydrolysis using the whole biofilm as a sensitive tool to evaluate the physiological state of immobilized bacterial cells

Abstract

Due to the increasing interest and the use of immobilized biocatalysts in bioremediation studies, there is a need for the development of an assay for quick and reliable measurements of their overall enzymatic activity. Fluorescein diacetate (FDA) hydrolysis is a widely used assay for measuring total enzymatic activity (TEA) in various environmental samples or in monoculture researches. However, standard FDA assays for TEA measurements in immobilized samples include performing an assay on cells detached from the carrier. This causes an error, because it is not possible to release all cells from the carrier without affecting their metabolic activity. In this study, we developed and optimized a procedure for TEA quantification in the whole biofilm formed on the carrier without disturbing it. The optimized method involves pre-incubation of immobilized carrier in phosphate buffer (pH 7.6) on the orbital shaker for 15 min, slow injection of FDA directly into the middle of the immobilized carrier, and incubation on the orbital shaker (130 rpm, 30◦C) for 1 h. Biofilm dry mass was obtained by comparing the dried weight of the immobilized carrier with that of the unimmobilized carrier. The improved protocol provides a simple, quick, and more reliable quantification of TEA during the development of immobilized biocatalysts compared to the original method.